ABSTRACT/SUMMARY A key effect of insulin is to increase glucose uptake into adipocytes by translocating the Glut4 glucose transporter from intracellular storage compartments to the plasma membrane (PM); a process essential for the postprandial blood glucose lower effect of insulin. Disruption of Glut4 trafficking contributes to hyperglycemia associated with insulin-resistance. In basal (unstimulated) adipocytes, Glut4 is predominantly excluded from the PM by a dynamic mechanism of rapid endocytosis and slow recycling. Insulin effects a net redistribution of Glut4 to the PM by accelerating exocytosis and inhibiting endocytosis. In both basal and insulin-stimulated conditions Glut4 is ferried to the PM by Glut4-specialized vesicles (GSVs). How insulin regulates the biogenesis and trafficking of GSVs to the PM are not known. The small GTPase, Rab10, is required for Glut4 translocation and in the past funding period we identified Sec16A as a Rab10 effector. Sec16A is known to function in the formation of ER COPII vesicles, and we have shown that its role in Glut4 trafficking is independent of that function. These data suggest that that Sec16A enhances GSV formation. In aim 1 of the project we will characterize the roles of Rab10-Sec16A GSV formation, adopting multiple complementary approaches that utilize biochemical, molecular cellular and microscopy methods to define GSV biogenesis, and to identify the Sec16A interacting proteins required for GSV formation. These studies will provide novel information significantly advancing our understanding of the biogenesis of GSVs and will thereby be revealing of a poorly understood step of insulin-regulation of Glut4 translocation. We have generated an adipose-specific Rab10 knockout mouse (aR10KO). The cellular phenotype of Rab10 KO primary adipocytes is identical to that of Rab10 knockdown in cultured adipocytes. Surprisingly, selective knockout of Rab10 in adipocytes induces insulin-resistance in the liver. Thus, adipocyte ablation of Rab10 alters adipocyte-liver communication to blunt liver insulin sensitivity. These data suggest that Rab10 KO alters the secreted adipokine profile (adipocyte hormones) in a way that impacts liver insulin sensitivity. In the aim 2 we define the mechanism underlying liver insulin resistance in aR10KO mice. We test the hypothesis that the ablation of Rab10 alters the secretion of adipokines, thereby affecting liver insulin sensitivity. We will investigate how the liver of aR10KO mice differ from control livers, use mass spectrometry coupled with cell-based functional assays to characterize the impact of Rab10 KO on the adipocyte secretome and to identify the adipokines responsible for altered liver metabolism. These studies will significantly advance our understanding of the endocrine role of adipocytes, and set the stage for future studies designed to elaborate the cellular mechanism by which adipocytes ?sense? changes in nutrients and how that information is relayed to alter secreted adipokines in response to changed nutrient status.